Charlotte Horsmans Poulsen

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides.

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory enzymes.

Antifouling enzymes and the biochemistry of marine settlement.

Antifouling coatings are used extensively on marine vessels and constructions, but unfortunately they are found to pose a threat to the marine environment, notably due to content of metal-based biocides. Enzymes have repeatedly been proposed as an alternative to traditional antifouling compounds. In this review, the enzymes claimed to hold antifouling activity are classified according to catalytic functions. The enzyme functions are juxtaposed with the current knowledge about the chemistry of settlement and adhesion of fouling organisms. Specific focus will be on bacteria, microalgae, invertebrate larvae and macroalgae zoospores. Two main concepts in enzyme-based antifouling are identified: breakdown of adhesive components and catalytic production of repellent compounds in-situ. The validity of the various modes of action is evaluated and the groups of enzymes with the highest potential are highlighted.

ROLES OF CHORISMATE MUTASE, ISOCHORISMATE SYNTHASE AND ANTHRANILATE SYNTHASE IN PLANTS

Abstract Chorismate is the substrate for a number of enzymes involved in the biosynthesis of aromatic compounds, making it a key branching point for secondary metabolites of the shikimate pathway. The enzymology and roles of three of the chorismate utilizing enzymes: chorismate mutase (EC 5.4.99.5), anthranilate synthase (EC 4.1.3.27) and isochorismate synthase (EC 5.4.99.6) are reviewed and discussed with emphasis of their importance for secondary metabolism. In particular the possible presence and localization of two isoenzymes, a regulated plastidic form and a non-regulated cytosolic form are discussed.

Purification and Characterization of a Hexose Oxidase with Excellent Strengthening Effects in Bread

ABSTRACT Hexose oxidase (EC 1.1.3.5) (HOX) was purified 51-fold from the red algae Chondrus crispus, by several chromatography methods, including hydrophobic interaction, chelating Sepharose, anion exchange, gel filtration, and chromatofocusing. Purified HOX was subjected to native PAGE and activity staining with nitroblue tetrazolium. For HOX electroeluted out of the gel and digested with endoproteinase Lys-C, the internal peptide sequence determined was: D-P-G-Y-I-V-I-D-V-N-A-G-T-(V or P)-D-K-P-D-P-X. The molecular mass, determined by gel filtration, was 126 kDa, versus 65 kDa determined by SDS-PAGE. The pI was determined to 4.64 and 4.79 as a double band on an isoelectrofocusing gel. Km was determined to 2.7 mM for D-glucose, 3.6 mM for D-galactose, 20.2 mM for cellobiose, 43.7 mM for maltose, 90.3 mM for lactose, 102 mM for xylose, and 531 mM for arabinose. The oxidation of thiol groups in gluten was determined by using Ellmans reagent: 5,5′-dithiobis (2-nitrobenzoic acid). The effect of HOX was comp...

Effects of elicitation on different metabolic pathways in Catharanthus roseus (L.)G.Don cell suspension cultures

Abstract Cell suspension cultures of Catharanthus roseus were elicited with an-autoclaved cell-free filtrate of Pythium aphanidermatum . The regulation of alkaloid, terpenoid, and phenolic biosynthetic pathways was studied by product formation and assay of the enzyme activity of anthranilate synthase (AS), tryptophan decarboxylase (TDC), strictosidine synthase (SSS), strictosidine-β-glucosidase (SG), isopentenyl diphosphate isomerase (IPP-isomerase), geraniol-10-hydroxylase (G10H), chorismate mutase (CM), and phenylalanine ammonia lyase (PAL). After elicitation, AS and TDC activities were induced which resulted in an increase in the amount of tryptamine in the cells. For SSS and SG, no significant induction was observed. Ajmalicine accumulation was not increased compared with that in controls. An increased amount of phenolic compounds was found in the culture medium, although CM activity was not induced and PAL activity was inhibited after elicitor treatment. The activities of the enzymes IPP-isomerase and G10H were inhibited after elicitation. Also, the incorporation of [ 14 C]IPP in terpenoid products such as squalene was inhibited. These findings indicate a limitation in the terpenoid pathway which could promote a shortage of secoiridoid precursors for terpenoid indole alkaloid biosynthesis.

Biomimetic silica encapsulation of enzymes for replacement of biocides in antifouling coatings.

Current antifouling technologies for ship hulls are based on metals such as cuprous oxide and co-biocides like zinc pyrithione. Due to the persistent adverse environmental effects of these biocides, enzyme-based antifouling paints are proposed as a bio-based, non-accumulating alternative. Here, a hydrogen peroxide-producing system composed of hexose oxidase (HOX, EC 1.1.3.5), glucoamylase (GA, EC 3.2.1.3) and starch is tested for the chemical and physical functionalities necessary for successful incorporation into a marine coating. The activity and stability of the enzymes in seawater was evaluated at different temperatures, and paint compatibility was assessed by measuring the distribution and activity of the enzymes incorporated into prototype coating formulations. We used a biomimetic encapsulation procedure for HOX through polyethylenimine-templated silica co-precipitation. The co-precipitation and formulation of a powder for mixing into a marine paint was performed in a one-step economical and gentle formulation process, in which silica co-precipitated HOX was combined with GA and starch to form the antifouling system. Silica co-precipitation significantly improved the stability and performance of the antifouling system in marine-like conditions. For example, encapsulation of HOX resulted in 46% higher activity at pH 8, and its stability in artificial seawater increased from retaining only 3.5% activity after 2 weeks to retaining 55% activity after 12 weeks. A coating comprising the full enzyme system released hydrogen peroxide at rates exceeding a target of 36 nmol cm−2 d−1 for 3 months in a laboratory assay, and had potential for prolonged action through incorporation in a self-polishing coating.

Characterization of the flavin association in hexose oxidase from Chondrus crispus

Hexose oxidase (EC 1.1.3.5) from Hansenula polymorpha was found to exhibit a dual covalent association of FAD with His79 via an 8α‐histidyl linkage as well as a covalent association between Cys138 and C‐6 of the isoalloxazine moiety of FAD. Spectral properties of the wild‐type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at 445 nm. An H79K mutant enzyme exhibited only one maximum at 437 nm. The difference absorption spectrum between an oxidized and a substrate‐reduced enzyme preparation showed maxima at 360 nm and 445 nm corresponding to an apparent novel type of association. Hexose oxidase showed a low, pH‐independent fluorescence at 525 nm when excited at 450 nm. Flavin was released from the holoenzyme by treatment with trypsin. Sequencing of the flavopeptide revealed two peptides comprising positions 74–91 and 132–157 associated with FAD in equimolar amounts. A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template. The model placed both of the sequences found above in the close vicinity of the FAD cofactor, and suggests covalent bonds between both His79 and Cys138 and FAD, in accordance with the chemical evidence. Based on the results, hexose oxidase is identified as incorporating FAD with a double covalent association with His79 and Cys138 in the holoenzyme. A reaction mechanism involving the concerted action of Tyr488 and Asp409 in hexose oxidase is suggested as the initiator of the proton abstraction from the substrate molecule in the active site.

Anthranilate synthase and chorismate mutase activities in transgenic tobacco plants overexpressing tryptophan decarboxylase fromCatharanthus roseus

TransgenicNicotiana tabacum L. ‘Petit Havana’ SR1 F1-plants expressing tryptophan decarboxylase cDNA (tdc) fromCatharanthus roseus (L.) G. Don under the control of the CaMV 35S promoter and terminator exhibited tryptophan decarboxylase (TDC) enzyme activity and accumulated tryptamine. The plants with the highest TDC activity contained 19 pkat per mg of protein. The influence of transgenic expression oftdc on the activities of anthranilate synthase (AS) and chorismate mutase (CM) were examined in 10 transgenic tobacco plants. The specific activities of these two chorismate-utilizing enzymes were not significantly affected by expression oftdc, despite their important functions as branch point enzymes in the shikimate pathway. The results indicate that the normal route of tryptophan biosynthesis in plants is sufficient to supply a considerable amount of this essential amino acid for the biosynthesis of secondary metabolites. Despite their increased tryptamine content, the growth and development of the transgenic tobacco plants expressingtdc appeared normal.

High-performance liquid chromatographic assay of anthranilate synthase from plant cell cultures

Abstract An assay is described for the enzyme anthranilate synthase (E.C. 4.1.3.27) from plant cell cultures, based on the fluorimetric detection of anthranilate after high-performance liquid chromatography on a LiChrosorb RP-8 Select B column. Depletion of the substrate chorismate and the presence of interfering enzymes can be followed by UV measurement. The rate of anthranilate formation was linear for at least 3 h at 30°C. The calibration graph was linear for at least 20 n M to 95 μ M . Anthranilate synthase was measured in Catharanthus roseus, Tabernaemontana divaricata, Cinchona robusta, Rubia tinctorum and Euonymus europaeus . The highest specific activity was found in C. roseus after induction for indole alkaloid production.

Implementation of cross-linked enzyme aggregates of proteases for marine paint applications

Cross-linked enzyme aggregates (CLEAs) of proteases were tested in artificial seawater (ASW) both as it is and as a component of the paint. It is found that all CLEAs have tolerance to xylene and have great stability in dried paint. Moreover, CLEA Bacillus licheniformis shows 900% activation during the storage in ASW in dried paint.